信号适体的设计策略及应用

信号适体兼具有分子识别和信号转导的功能.从随机寡核苷酸库中筛选出的适体,要经过合理设计和筛选后修饰,才具备信号转导功能.信号适体可分为标记和非标记两大类.本文着重介绍荧光标记信号适体的设计策略,包括基于荧光偏振分析标记一个荧光基团,及基于荧光共振能量转移同时标记荧光基团、淬灭基团,或两个荧光基团的信号适体(包括分子信标适体、结构转换和原位标记信号适体).非标记信号适体的设计,有嵌合法、置换法、光转换复合物法,及适体－多聚物偶联法.此外,亦可直接从体外筛选出信号适体.信号适体的诸多优点利于其用于生物传感器及均相液相中实时蛋白识别与定量分析.     

功能核酸荧光免标记型定量统一化检测技术的研究进展

功能核酸是一类具有特定空间构象、执行特异生物功能的天然或者人工核酸序列,具有易于修饰、价格低廉、稳定性高、特异性强等优势,其搭载免标记荧光传感系统后,功能核酸可起到将多种靶物质统一转为较为稳定的核酸信号,以及通过核酸扩增对检测信号进行扩增等重要作用。而免标记荧光传感技术可以在检测中免去荧光标记时荧光、猝灭基团的筛选和标记过程的繁琐和成本,同时可保证与核酸的特异或非特异性结合后产生荧光信号变化。通过两种技术的优势结合,检测的灵敏度、实时性可进一步提高,逐渐被广泛应用于环境污染物检测、食品风险因子检测、疾病诊断等多个领域。首先明确功能核酸与荧光定量检测技术等相关概念,详细阐述了几种重要的荧光物质的特点以及其与功能核酸的分子识别、作用方式与发光机制,接着紧紧围绕该种传感技术的几个特点,从功能核酸荧光免标记型定量统一化检测技术与其实际应用角度进行分类介绍与评价对比,最后就功能核酸荧光免标记型定量统一化检测技术在多种领域的检测分析中的研究意义以及存在的问题进行讨论,并对未来的发展与应用作出展望。     

荧光纳米生物传感器研究进展

荧光纳米生物传感器检测物质具有灵敏度高、响应迅速、抗干扰性强、无需参比电极等特点而被广泛地运用于生物传感技术领域。本文综述了荧光纳米生物传感器种类和特点,介绍了国内外近期在荧光纳米生物传感器及在生物检测方面的一些研究成果及进展,并作了分析比较。着重讨论了纳米粒子荧光生物传感器和光纤纳米荧光生物传感器的特性及其在生物分析中的应用。     

功能核酸荧光标记型定量统一化检测技术的研究进展

功能核酸是一类具有特定空间构象、执行特异生物功能的天然或者人工核酸序列,具有易于修饰、价格低廉、稳定性高、特异性强等优势,搭载荧光传感系统,组装成多种荧光生物传感器,被广泛应用于环境污染物检测、食品风险因子检测、疾病诊断等多个领域。首先明确功能核酸与荧光定量检测技术等相关概念,详细阐述了几种重要的荧光物质的特点以及其与功能核酸的分子识别、作用方式与发光机制,再主要从功能核酸荧光标记型定量统一化检测技术与其实际应用角度进行分类介绍与评价对比,最后就功能核酸荧光标记型定量统一化检测技术在多种领域的检测分析中的研究意义以及存在的问题进行讨论,并对未来的发展与应用作出展望。     

基于适体的石英晶体微天平传感器的研究进展

适体(Aptamer)是通过指数富集配体系统进化技术(Systematic evolution of ligands by exponential enrichment,SELEX)从人工合成的随机单链寡核苷酸文库里筛选出来的短链寡核苷酸序列,具有分子量小、结构简单、易进行修饰、靶标范围广泛,并且与靶标分子之间具有高特异性和高亲和力等特点。相应地,适体的这些独特的性质可用于制备各种不同的传感器,根据各种传感器的不同原理,本文着重概述了常用于检测适体与靶标之间亲和力的电化学生物传感器、光学生物传感器和压电晶体传感器。这3种方法的检测都具有检测时间短和检测限低的优势。其中压电晶体传感器又称石英晶体微天平(Quartz crystal microbalance,QCM),除了在SELEX技术中可应用于表征候选适体的靶向能力外,还可用于构建高灵敏度和高特异性的适体石英晶体传感器。简要介绍了基于适体的石英晶体微天平传感器基本原理,对近年来QCM在表征和检测适体与其靶标,包括小分子、离子、蛋白质、细胞、细菌和病毒等物质相互作用的研究现状进行综述,总结分析了QCM技术的优缺点。旨在为适体的筛选以及适体在基础研究、临床诊断和疾病治疗中的进一步应用提供参考。     

肽适体的筛选及其应用

近年来,生物传感器得到了长足发展,其中适配体的研究起到了重要推动作用.肽适体是可与靶标物质特异性结合的短肽.受制于筛选、合成以及纯化方法,肽适体的发展目前落后于核酸适配体,但是肽适体的高亲和力、强特异性、良好的生物亲和性等系列优越性,让肽适体具有巨大应用前景.肽适体的筛选获得可通过多种方式完成,除了传统的酵母双杂交、噬菌体展示、核糖体展示等技术,新兴的基于生物信息学的分子对接预测等技术更是加速了肽适体的发展.肽适体不仅可直接应用于临床医疗,还可设计成生物传感器广泛应用于精准营养、环境监测以及新型材料识别等领域.本文旨在对肽适体筛选及其应用做全面的梳理.     

场效应晶体管生物传感器在生物医学检测中的应用研究进展*

场效应晶体管生物传感器因其灵敏度高、分析速度快、无标记、体积小、操作简单等特点而受到了很多关注,广泛应用于DNA、蛋白质、细胞、离子等生物识别物的检测。近年来,更有纳米材料和微电子技术在传感器设计中提高传感器的传感性能,场效应晶体管生物传感器朝着高灵敏、微型化、快速化以及多功能化的方向以令人惊叹的速度发展。研究场效应晶体管生物传感器工作原理,阐述近年来场效应晶体管生物传感器在生物医学检测领域中最新的研究进展与应用,探讨场效应晶体管生物传感器克服各种缺陷的应对策略,为该传感器在未来生物医学检测中的开发提供参考。     

基于信号放大技术的适体生物传感器研究进展

信号放大技术因其能实现低浓度分子检测,灵敏度高而在多个研究领域发展非常迅速。而适体作为识别分子已成功应用于多种生物传感器平台,在医疗诊断、环境检测、生化分析中显示出良好的应用前景。近年来,以适体为识别元件的生物传感器越来越受到人们的关注。综述了近3年来基于信号放大技术的适体生物传感器研究新发展。     

荧光铜纳米簇介导的生物传感器的研究进展

荧光铜纳米簇(Fluorescent copper nanoclusters,CuNCs)是以脱氧核糖核酸链(Deoxyribonucleic acid,DNA)为模板,以二价铜离子(Cu2+)、抗坏血酸等为反应物生成的铜晶体,纳米级大小,其具有荧光性,可以作为生物传感器输出信号的一种方式。荧光铜纳米簇的生成快速、简便、安全,因此近年来涌现出很多关于荧光铜纳米簇原理和应用方面的研究。从支持传感器工作的介导物质以及信号输出方式两方面对荧光铜纳米簇进行分类,详细阐述了每一类别传感器工作的原理,并对比同类型传感器的优缺点,最后对荧光铜纳米簇介导的生物传感器目前存在的不足及今后的发展趋势进行了展望。以便读者对荧光铜纳米簇生物传感器发展历程和方向,对荧光铜纳米簇生物传感器的实用性和多形性有所了解,在未来的研究发展中得到启示,使荧光铜纳米簇成为一种更加实用和便捷的生物传感工具。     

基于发光共轭聚合物的核酸生物传感器

本文介绍了以发光共轭聚合物为基础,以荧光为检测手段,针对核酸进行特异性识别的一类检测信号放大的生物传感器。主要介绍了DNA和RNA的几种检测方法,探讨了影响荧光传感信号的因素。     

Analytical potential of gold nanoparticles in functional aptamer-based biosensors

Gold nanoparticles (AuNPs) exhibit many predominant capabilities such as high biocompatibility, chemical stability, strong localized surface plasmon resonance absorption, and high extinction coefficient in the visible region. These properties have enabled the extensive use of AuNPs in optical and electrochemical biosensors. As a kind of functional nucleic acids, aptamers can be considered as a valid alternative to antibodies or other bio-receptors and have been widely employed to develop novel biosensors. We are summarizing here the state of the art of AuNP-based biosensors that use functional aptamers as molecular recognition elements. In many cases, AuNPs themselves can be used as a probe for detection, such as various colorimetric aptasensors and fluorescent aptasensors. They also can be used as probe vectors to enlarge detection signals and to increase the number of conceivable substrates in electrochemical aptasensors.     

G-quadruplex-forming aptamer enhances the peroxidase activity of myoglobin against luminol

Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin''s peroxidase activity was discovered. Through our strategy—in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.     

Progress of new label-free techniques for biosensors: a review

The detection techniques used in biosensors can be broadly classified into label-based and label-free. Label-based detection relies on the specific properties of labels for detecting a particular target. In contrast, label-free detection is suitable for the target molecules that are not labeled or the screening of analytes which are not easy to tag. Also, more types of label-free biosensors have emerged with developments in biotechnology. The latest developed techniques in label-free biosensors, such as field-effect transistors-based biosensors including carbon nanotube field-effect transistor biosensors, graphene field-effect transistor biosensors and silicon nanowire field-effect transistor biosensors, magnetoelastic biosensors, optical-based biosensors, surface stress-based biosensors and other type of biosensors based on the nanotechnology are discussed. The sensing principles, configurations, sensing performance, applications, advantages and restriction of different label-free based biosensors are considered and discussed in this review. Most concepts included in this survey could certainly be applied to the development of this kind of biosensor in the future.     

Platelet-derived growth factor oncoprotein detection using three-dimensional carbon microarrays

The potential of aptamers as ligand binding molecule has opened new avenues in the development of biosensors for cancer oncoproteins. In this paper, a label-free detection strategy using signaling aptamer/protein binding complex for platelet-derived growth factor (PDGF-BB) oncoprotein detection is reported. The detection mechanism is based on the release of fluorophore (TOTO intercalating dye) from the target binding aptamer's stem structure when it captures PDGF. Amino-terminated three-dimensional carbon microarrays fabricated by pyrolyzing patterned photoresist were used as a detection platform. The sensor showed near linear relationship between the relative fluorescence difference and protein concentration even in the sub-nanomolar range with an excellent detection limit of 5pmol. This detection strategy is promising in a wide range of applications in the detection of cancer biomarkers and other proteins.     

Electrochemical-based biosensors for detection of Mycobacterium tuberculosis and tuberculosis biomarkers

Abstract

Early detection of tuberculosis (TB) reduces the interval between infection and the beginning of treatment. However, commercially available tests cannot discriminate between BCG-vaccinated healthy persons and patients. Also, they are not suitable to be used for immunocompromised persons. In recent years, biosensors have attracted great attention due to their simple utility, accessibility, and real-time outputs. These sensors are increasingly being considered as pioneering tools for point-of-care diagnostics in communities with a high burden of TB and limited accessibility to reference laboratories. Among other types of biosensors, the electrochemical sensors have the advantages of low-cost operation, fast processing, simultaneous multi-analyte analyzing, operating with turbid samples, comparable sensitivity and readily available miniaturization. Electrochemical biosensors are sub-divided into several categories including: amperometric, impedimetric, potentiometric, and conductometric biosensors. The biorecognition element in electrochemical biosensors is usually based on antibodies (immunosensors), DNAs or PNAs (genosensors), and aptamers (aptasensors). In either case, whether an interaction of the antigen–antibody/aptamer or the hybridization of probe with target mycobacterial DNA is detected, a change in the electrical current occurs that is recorded and displayed as a plot. Therefore, impedimetric-based methods evaluate resistance to electron transfer toward an electrode by a Nyquist plot and amperometric/voltammetric-based methods weigh the electrical current by means of cyclic voltammetry, square wave voltammetry, and differential pulse voltammetry. Electrochemical biosensors provide a promising scope for the new era of diagnostics. As a consequence, they can improve detection of Mycobacterium tuberculosis traces even in attomolar scales.     

Nanocrystalline diamond impedimetric aptasensor for the label-free detection of human IgE

Like antibodies, aptamers are highly valuable as bioreceptor molecules for protein biomarkers because of their excellent selectivity, specificity and stability. The integration of aptamers with semiconducting materials offers great potential for the development of reliable aptasensors. In this paper we present an aptamer-based impedimetric biosensor using a nanocrystalline diamond (NCD) film as a working electrode for the direct and label-free detection of human immunoglobulin E (IgE). Amino (NH(2))-terminated IgE aptamers were covalently attached to carboxyl (COOH)-modified NCD surfaces using carbodiimide chemistry. Electrochemical impedance spectroscopy (EIS) was applied to measure the changes in interfacial electrical properties that arise when the aptamer-functionalized diamond surface was exposed to IgE solutions. During incubation, the formation of aptamer-IgE complexes caused a significant change in the capacitance of the double-layer, in good correspondence with the IgE concentration. The linear dynamic range of IgE detection was from 0.03 μg/mL to 42.8 μg/mL. The detection limit of the aptasensor reached physiologically relevant concentrations (0.03 μg/mL). The NCD-based aptasensor was demonstrated to be highly selective even in the presence of a large excess of IgG. In addition, the aptasensor provided reproducible signals during six regeneration cycles. The impedimetric aptasensor was successfully tested on human serum samples, which opens up the potential of using EIS for direct and label-free detection of IgE levels in blood serum.     

Unraveling the binding mode of a methamphetamine aptamer: A spectroscopic and calorimetric study

Nucleic-acid aptamers are bio-molecular recognition agents that bind to their targets with high specificity and affinity and hold promise in a range of biosensor and therapeutic applications. In the case of small-molecule targets, their small size and limited number of functional groups constitute challenges for their detection by aptamer-based biosensors because bio-recognition events may both be weak and produce poorly transduced signals. The binding affinity is principally used to characterize aptamer-ligand interactions; however, a structural understanding of bio-recognition is arguably more valuable in order to design a strong response in biosensor applications. Using a combination of nuclear magnetic resonance, circular dichroism, and isothermal titration calorimetry, we propose a binding model for a new methamphetamine aptamer and determine the main interactions driving complex formation. These measurements reveal only modest structural changes to the aptamer upon binding and are consistent with a conformational-selection binding model. The aptamer-methamphetamine complex formation was observed to be entropically driven, apparently involving hydrophobic and electrostatic interactions. Taken together, our results exemplify a means of elucidating small molecule-aptamer binding interactions, which may be decisive in the development of aptasensors and therapeutics and may contribute to a deeper understanding of interactions driving aptamer selection.     

Molecular signaling aptamers for real-time fluorescence analysis of protein

Aptamers are a new class of nucleic acids that are selected in vitro for binding target molecules with high affinity and selectivity. They are promising protein-binding molecular probes that rival conventional antibodies for protein analysis. There have been recent advances in the development of molecular signaling aptamers that can transduce target protein binding to sensitive fluorescence signal changes. This facilitates the real time protein monitoring in homogenous solution as well as potentially in vivo. Different signaling strategies of using dual labeled aptamers based on fluorescence resonance energy transfer (FRET), one fluorophore labeled aptamers based on fluorescence anisotropy assay, or other label-free aptamers are reviewed.     

Aptasensors for biosecurity applications

Nucleic acid (aptasensors) have found steadily increased utility and application over the past decade. In particular, aptamers have been touted as a valuable complement to and, in some cases, replacement for antibodies owing to their structural and functional robustness as well as their ease in generation and synthesis. They are thus attractive for biosecurity applications (e.g. pathogen detection) and are especially well suited because their in vitro generation process does not require infection of any host systems. Herein we provide a brief overview of the aptamers generated against pathogens and toxins over the past few years. In addition, a few recently described detection platforms using aptamers and potentially suitable applications for biosecurity will be discussed.